Stable hemoglobin mutants and uses thereof

Abstract

The application discloses a hemoglobin mutant with improved stability and application thereof, and belongs to the technical field of genetic engineering. The application lays a foundation for obtaining an ideal stable human hemoglobin mutant by designing a high-throughput screening method for the structural stability of human hemoglobin and the hematin binding intensification mutant. The application determines hot amino acid sites for the structural stability and thermal stability modification of human hemoglobin through semi-rational design, and further carries out iterative saturation mutation to obtain an ideal mutant rHb 9th The application has the advantages that in terms of structural stability, thermal stability, anti-autoxidation capacity, hematin binding rate, anti-hematin loss rate and human hemoglobin yield, etc. wild‑type The application has the advantages that in terms of structural stability, thermal stability, anti-autoxidation capacity, hematin binding rate, anti-hematin loss rate and human hemoglobin yield, etc.

Description

TECHNICAL FIELD

[0001] The present application relates to hemoglobin mutants with improved stability and applications thereof, and belongs to the technical field of genetic engineering. BACKGROUND

[0002] Human hemoglobin is a tetrameric protein, which includes two identical alpha globins (141 residues) and two identical beta globins (146 residues). Each globin binds one molecule of hemoglobin, which further carries one molecule of oxygen. Hemoglobin plays a crucial role in oxygen transport, which is more than 2 times more efficient than red blood cells. Currently, compared with the traditional method of extracting hemoglobin from human blood, heterologous synthesis of recombinant human hemoglobin (rHb) in Escherichia coli or Saccharomyces cerevisiae has many advantages, including reducing the risk of disease transmission and saving costs. However, the synthesis of rHb lacks the antioxidant system (superoxide dismutase and catalase) present in red blood cells, which leads to the instability of rHb, further affecting its application as an artificial oxygen carrier.

[0003] The instability of rHb mainly includes protein structural instability and rapid loss of the cofactor heme due to rapid autoxidation of rHb. Protein structural instability can lead to human hemoglobin being easily decomposed into dimers and monomers, causing rHb degradation and causing serious damage to the kidneys. Rapid loss of heme can cause rHb to lose its ability to transport oxygen and accelerate the degradation of rHb. Currently, the methods for strengthening the stability of rHb mainly include chemical modification and genetic engineering mutagenesis. However, chemical modification not only increases the cost of rHb synthesis, but also is not conducive to its subsequent large-scale application. And the genetic engineering mutagenesis strategy for rHb stability modification only relies on rational design of a few sites and other strategies, and the efficiency of modification is limited.

[0004] Therefore, in order to obtain more stable rHb mutants, random mutation, directed evolution or rational modification of neglected key residues can be used to generate a large number of potential mutants. Then, high-throughput screening based on improved fluorescent biosensors can quickly obtain ideal rHb mutants. SUMMARY

[0005] The present application first designs a high-throughput screening strategy for human hemoglobin mutants with improved structural stability and heme binding, and simultaneously determines the corresponding hot amino acid sites for modification using a semi-rational design strategy, thereby realizing the gradual strengthening of the structural stability and heme binding capacity of human hemoglobin.

[0006] The present application provides a hemoglobin mutant, which has the mutation shown in (a) or (b) based on the starting sequence:

[0007] (a) the 25th glycine of alpha globin is mutated to cysteine, the 57th glycine is mutated to lysine, and the 114th proline is mutated to isoleucine; the 2nd histidine of beta globin is mutated to glutamic acid, the 97th histidine is mutated to tryptophan, and the 102nd asparagine is mutated to threonine; the obtained mutant is named as alpha-G25C / G57K / P114I, beta-H2E / H97W / N102T;

[0008] (b) the 25th glycine of alpha globin is mutated to cysteine, the 42nd tyrosine is mutated to serine, the 57th glycine is mutated to lysine, and the 114th proline is mutated to isoleucine; the 2nd histidine of beta globin is mutated to glutamic acid, the 44th serine is mutated to aspartic acid, the 57th glycine is mutated to lysine, the 97th histidine is mutated to tryptophan, and the 102nd asparagine is mutated to threonine; the obtained mutant is named as alpha-G25C / Y42S / G57K / P114I, beta-H2E / S44D / P51N / H97W / N102T.

[0009] In an embodiment, the starting sequence of alpha globin is shown as SEQ ID NO. 1; the starting sequence of beta globin is shown as SEQ ID NO. 2.

[0010] In an embodiment, the amino acid sequence of the mutant alpha-G25C / G57K / P114I, beta-H2E / H97W / N102T is shown as SEQ ID NO. 3 and SEQ ID NO. 4.

[0011] In an embodiment, the amino acid sequence of the mutant alpha-G25C / Y42S / G57K / P114I, beta-H2E / S44D / P51N / H97W / N102T is shown as SEQ ID NO. 5 and SEQ ID NO. 6.

[0012] The present application also provides a gene encoding the hemoglobin mutant.

[0013] The present application also provides a recombinant plasmid carrying the gene.

[0014] The present application also provides a recombinant microorganism expressing the hemoglobin mutant.

[0015] The present application also provides a biosensor that can be used for screening structure-stable hemoglobin, which has a sequence with the structure shown as “alpha globin-(SSG)2-CysG A -(G4S)3-beta globin”; wherein CysG Ais a siroheme synthase encoded by the sequence shown in SEQ ID NO. 8, (SSG)2 is a linker peptide SSGSSG, and (G4S)3 is a linker peptide GGGGSGGGGSGGGGS.

[0016] In an embodiment, the biosensor is constructed on a plasmid, including but not limited to pETDuet-1.

[0017] In an embodiment, the E. coli starting strain required for the rHb structure stability modification includes but is not limited to E. coli BL21 (DE3).

[0018] The present application also provides the use of the recombinant microorganism in the preparation of the hemoglobin.

[0019] In an embodiment, the use is to culture the recombinant microorganism in a culture medium, and adding hemin during the culture process.

[0020] In an embodiment, the concentration of the hemin in the culture environment is 0-20 mg / L.

[0021] The present application provides a high-throughput screening method for a human hemoglobin hemin binding enhanced mutant, which is to introduce a double-plasmid screening system into the cell of an E. coli starting strain; the double-plasmid includes a recombinant plasmid pTf16-HS1 M7A carrying a hemin detection element HS1 M7A and a recombinant plasmid pET-rHb carrying a hemoglobin coding gene.

[0022] In an embodiment, the amino acid sequence of the human hemoglobin rHb wild-type is shown in PDB: 2DN2, the PDB ID of red fluorescent protein mKATE2 is 3PJ5, and the PDB ID of green fluorescent protein eGFP is 2GX2.

[0023] In an embodiment, the sequence of the hemin detection element HS1 M7A is shown in SEQ ID NO. 7.

[0024] In an embodiment, the E. coli starting strain includes but is not limited to E. coli R11-HEME, which has been disclosed in the paper Whole-cell P450 biocatalysis using engineered with fine-tuned hemebiosynthesis.

[0025] In an embodiment, the double-plasmid screening system determined by the high-throughput screening strategy for the rHb hemin binding enhanced modification is rHb-HS1 M7A(RSF ori). Wherein, the pETDuet-1 plasmid is screened by positive clones of ampicillin resistance, the replicon is pBR322 ori, and the rHb coding gene is fused downstream of the T7 promoter, and the expression of rHb is induced by isopropyl-beta-D-thiogalactoside (IPTG). The pTf16 (RSF ori) plasmid is screened by positive clones of chloramphenicol resistance, the replicon is RSF ori, and the HS1 M7A coding gene is fused downstream of the araB promoter, and the expression of HS1 M7A is induced by arabinose.

[0026] The present application provides a method for improving the structural stability of human hemoglobin, which is to mutate one or more of the following amino acid sites of human hemoglobin: α-G15, α-K16, α-G25, α-G57, α-V93, α-D94, α-P114, β-H2, β-G16, β-G69, β-S72, β-H97 and β-N102.

[0027] In one embodiment, the method is to mutate the 25th glycine of α-globin to cysteine, mutate the 57th glycine to lysine, and mutate the 114th proline to isoleucine; mutate the 2nd histidine of β-globin to glutamic acid, mutate the 97th histidine to tryptophan, and mutate the 102nd asparagine to threonine; obtain mutant α-G25C / G57K / P114I, β-H2E / H97W / N102T.

[0028] The present application also provides the use of the hemoglobin mutant and the recombinant microorganism in the preparation of hemoglobin or its related products.

[0029] Beneficial effects:

[0030] (1) The present application designs a high-throughput screening strategy for the structural stability and heme binding capacity of the multi-subunit protein human hemoglobin, which is beneficial to the efficient acquisition of ideal mutants.

[0031] (2) The present application uses a semi-rational design strategy to determine the key amino acid sites of the iterative saturation library for the structural stability and heme binding capacity of human hemoglobin, and the obtained mutant rHb 9th has significantly improved thermal stability (TM value increased by 4.2℃), structural stability (increased by 26.7%), autoxidation capacity (decreased by 15.4 times), heme binding rate (increased by 37.4%), heme loss rate (decreased by 15.5 times and 2.3 times in α and β globin respectively), and protein yield (increased by 1.63 times) compared with rHb wild-type . BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 High-throughput screening strategy for the stability engineering of human hemoglobin. Wherein, a is the determination of appropriate high-throughput screening strategy by linker optimization. b is the rHb-CysG of different linkers A CysG of fusion protein A Red fluorescence intensity. c is rHb-CysG A Correlation analysis of fluorescence intensity of fusion protein and mutation energy of mutant. Red line represents (G4S)3 linker connecting the C-terminal of β-globin and the C-terminal of CysG A Correlation analysis of fluorescence intensity of fusion protein and mutation energy of mutant. Red line represents (G4S)3 linker connecting the C-terminal of β-globin and the C-terminal of CysG A Correlation analysis of fluorescence intensity of fusion protein and mutation energy of mutant. Red line represents (G4S)3 linker connecting the C-terminal of β-globin and the C-terminal of CysG

[0033] Figure 2 High-throughput screening strategy for the heme binding enhancement engineering of human hemoglobin. Wherein, a is the construction of high-throughput screening strategy of human hemoglobin variants (PDB ID: 2DN2 of human hemoglobin, PDB ID: 3PJ5 of red fluorescent protein mKATE2, PDB ID: 2GX2 of green fluorescent protein eGFP). b is the eGFP / mKATE2 fluorescence of rHb-HS1 system under different replicons (RSF ori and p15A ori) or inducible conditions M7A eGFP / mKATE2 fluorescence of rHb-HS1 system. 1, 2 represent adding inducer IPTG first, then adding arabinose. 3 represents adding two kinds of inducers (IPTG and arabinose) at the same time. c is the verification of the rate of heme loss in human hemoglobin by adding different heme dissociation reagents. d is the fluorescence ratio of rHb-HS1 M7A Correlation analysis of fluorescence ratio of rHb-HS1 and mutation energy of rHb mutant.

[0034] Figure 3 Rational design and engineering of human hemoglobin to improve its structural stability. Wherein, a is the determination of mutation sites to improve the stability of rHb by rational design. Green amino acids represent candidate sites after virtual saturation mutation, and red amino acids represent the final sites used for iterative saturation mutation. b is the process of iterative saturation mutation. c-g are the properties of ideal mutants determined by each round of iterative saturation mutation: c is fluorescence intensity, d is biomass and human hemoglobin yield, e is the secondary structure of the mutant determined by circular dichroism spectrum, f is the thermal stability of the mutant determined by differential scanning calorimetry, and g is the autoxidation rate of human hemoglobin mutant.

[0035] Figure 4Rational design and engineering of human hemoglobin to improve its heme binding ability. a is the selection of appropriate mutation sites to enhance the heme binding strength of hemoglobin. Black amino acids represent the sites finally used for iterative saturation mutagenesis. b is the process of iterative saturation mutagenesis. c-g are the properties of the ideal mutant determined by each round of iterative saturation mutagenesis: c is the eGFP / mKATE2 fluorescence ratio, d is the CysG A fluorescence intensity, e is the biomass and human hemoglobin yield, f is the heme loss rate, and g is the heme binding rate. DETAILED DESCRIPTION

[0036] Purification and quantification of human hemoglobin: The synthesized rHb was suspended in 20.0 mM phosphate buffer (PBS, including 5.0 mM NaH2PO4, 48.0 mM Na2HPO4, 0.5 M NaCl, pH 7.4) and 1 mM benzylsulfonyl fluoride was added as a protease inhibitor to prevent rHb degradation at a final concentration. The cells were broken at 4 °C using a high-pressure homogenizer (Union Biotech). The supernatant was collected by centrifugation at 4 °C for 10 minutes (7000 rpm) and added to a His-tagged affinity gravity column, which was washed with 10 column volumes of PBS buffer A (pH 7.4, final concentration of 20.0 mM imidazole), then 10 column volumes of PBS buffer B (pH 7.4, final concentration of 80.0 mM imidazole) to wash away other proteins, and rHb was eluted with PBS buffer C (pH 7.4, 0.5 M imidazole). The purified rHb was then concentrated using an Amicon Ultra-0.5 centrifuge (Merck KGaA) to reduce the imidazole concentration to below 50.0 mM, and the protein concentration was determined using a Bradford protein detection kit (Beyotime Biotech). The quality of the purified rHb was verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Ultra-0.5 centrifuge (Merck KGaA) to reduce the imidazole concentration to below 50.0 mM, and the protein concentration was determined using a Bradford protein detection kit (Beyotime Biotech). The quality of the purified rHb was verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

[0037] Determination of oxygen saturation: Sodium dithionite was used as a deoxygenating agent and reducing agent for methemoglobin. rHb and sodium dithionite solution were mixed in a 1:13 ratio in a 96-well black plate, and then the rHb was converted to deoxyhemoglobin. Next, the mixed solution was incubated at 37 °C, and the absorbance values of A 556 and A 523 were determined by a microplate reader (BioTek synergy H1). Between each measurement, the 96-well black plate was shaken at a speed of 300 rpm / min for 1 minute. The method for determining the oxygen saturation of rHb followed the previous method:

[0038] Determination of oxygen saturation: Sodium dithionite was used as a deoxygenating agent and reducing agent for methemoglobin. rHb and sodium dithionite solution were mixed in a 1:13 ratio in a 96-well black plate, and then the rHb was converted to deoxyhemoglobin. Next, the mixed solution was incubated at 37 °C, and the absorbance values of A 556 and A 523 were determined by a microplate reader (BioTek synergy H1). Between each measurement, the 96-well black plate was shaken at a speed of 300 rpm / min for 1 minute. The method for determining the oxygen saturation of rHb followed the previous method:

[0038] Determination of oxygen saturation: Sodium dithionite was used as a deoxygenating agent and reducing agent for methemoglobin. rHb and sodium dithionite solution were mixed in a 1:13 ratio in a 96-well black plate, and then the rHb was converted to deoxyhemoglobin. Next, the mixed solution was incubated at 37 °C, and the absorbance values of A 556 and A 523 were determined by a microplate reader (BioTek synergy H1). Between each measurement, the 96-well black plate was shaken at a speed of 300 rpm / min for 1 minute. The method for determining the oxygen saturation of rHb followed the previous method:

[0038] Determination of oxygen saturation: Sodium dithionite was used as a deoxygenating agent and reducing agent for methemoglobin. rHb and sodium dithionite solution were mixed in a 1:13 ratio in a 96-well black plate, and then the rHb was converted to deoxyhemoglobin. Next, the mixed solution was incubated at 37 °C, and the absorbance values of A 556 and A 523 were determined by a microplate reader (BioTek synergy H1). Between each measurement, the 96-well black plate was shaken at a speed of 300 rpm / min for 1 minute. The method for determining the oxygen saturation of rHb followed the previous method:

[0038] Determination of oxygen saturation: Sodium dithionite was used as a deoxygenating agent and reducing agent for methemoglobin. rHb and sodium dithionite solution were mixed in a 1:13 ratio in a 96-well black plate, and then the rHb was converted to deoxyhemoglobin. Next, the mixed solution was incubated at 37 °C, and the absorbance values of A 556 and A 523 were determined by a microplate reader (BioTek synergy H1). Between each measurement, the 96-well black plate was shaken at a speed of 300 rpm / min for 1 minute. The method for determining the oxygen saturation of rHb followed the previous method:

[0038] Determination of oxygen saturation: Sodium dithionite was used as a deoxygenating agent and reducing agent for methemoglobin. rHb and sodium dithionite solution were mixed in a 1:13 ratio in a 96-well black plate, and then the rHb was converted to deoxyhemoglobin. Next, the mixed solution was incubated at 37 °C, and the absorbance values of A 556 and A 523 were determined by a microplate reader (BioTek synergy H1). Between each measurement, the 96-well black plate was shaken at a speed of 300 rpm / min for 1 minute. The method for determining the oxygen saturation of rHb followed the previous method:

[0038] Determination of oxygen saturation: Sodium dithionite was used as a deoxygenating agent and reducing agent for methemoglobin. rHb and sodium dithionite solution were mixed in a 1:13 ratio in a 96-well black plate, and then the rHb was converted to deoxyhemoglobin. Next, the mixed solution was incubated at 37 °C, and the absorbance values of A 556 and A 523 were determined by a microplate reader (BioTek synergy H1). Between each measurement, the 96-well black plate was shaken at a speed of 300 rpm / min for 1 minute. The method for determining the oxygen saturation of rHb followed the previous method:

[0038] Determination of oxygen saturation: Sodium dithionite was used as a deoxygenating agent and reducing agent for methemoglobin. rHb and sodium dithionite solution were mixed in a 1:13 ratio in a 96-well black plate, and then the rHb was converted to deoxyhemoglobin. Next, the mixed solution was incubated at 37 °C, and the absorbance values of A 556 and A 523 were determined by a microplate reader (BioTek synergy H1). Between each measurement, the 96-well black plate was shaken at a speed of 300 rpm / min for 1 minute. The method for determining the oxygen saturation of rHb followed the previous method:

[0038] Determination of oxygen saturation: Sodium dithionite was used as

[0039] K Hb A is the absorbance of rHb when it is fully deoxygenated; K HbO2 B is the absorbance of rHb when it is fully oxygenated; K x C is the absorbance of rHb when oxygenation is carried out to a specific time (t).

[0040] Oxygen equilibrium curve: The oxygen equilibrium curve of rHb was determined using an oxygen binding / release instrument (BL00DOX-2018), and the preparation of ferrohemoglobin was performed using a 0.1 M sodium hyposulfite solution. During the measurement process, the partial pressure of both air and nitrogen was set to 0.1 MPa, and the reaction temperature was maintained at 37 °C. 3.0 mg of rHb was mixed with 4.0 mL of a buffer solution (including 50.0 mM HEPES, 0.1 M NaCl, 0.1 mM EDTA, and 0.1 mM bovine serum albumin) and injected into the sample cell of the oxygen binding / release instrument. The air valve was opened to allow air to be introduced at a constant rate (13.0 mL / min) until the P O2 value was stable. Subsequently, nitrogen was introduced at a constant rate (13.0 mL / min) to measure the oxygen dissociation rate until the P O2 value was stable again, thereby calculating the P 50 value and Hill coefficient of rHb.

[0041] Circular dichroism spectrum for detecting the structural stability of rHb: Circular dichroism measurement was performed using a Chirascan spectropolarimeter. 6.0 M guanidine hydrochloride (GdnHCl) was prepared in a 0.2 M potassium phosphate buffer (containing 68.0 mM KH2PO4 and 131.0 mM K2HPO4, pH 7.0), and the pH value of the potassium phosphate buffer was adjusted to 7.0 after the addition of the denaturant GdnHCl. 12.0 mM rHb and different concentrations of GdnHCl (0.5, 1.0, 1.5, 1.7, 1.8, 1.9, 2.0, 2.5, 3.0, and 4.0 M) were dissolved in 0.2 M potassium phosphate buffer (pH 7.0) to prepare a mixture. The rHb-GdnHCl mixture was incubated in a 10 °C water bath for 1 hour before the spectrum measurement. The circular dichroism spectrum of each sample was recorded in the range of 190 to 250 nm using a JASCO 1700 CD spectropolarimeter, and the negative ellipticity change at 222 nm was monitored to characterize the secondary structure change of rHb.

[0042] Differential scanning calorimetry for measuring the thermal stability of rHb: The thermal denaturation process of rHb was studied using nano-DSC in the temperature range of 40-90 °C. 0.3 mL of rHb (15.5 mM) was loaded into the sample cell, and the differential scanning curve of the degassed sample (equilibrium sample for 600 seconds) was recorded at a scanning rate of 1 °C per minute under a constant external pressure of 3 atm. The P wild-typeAs a control, the data were analyzed using a Nano analyzer.

[0043] Determination of the auto-oxidation rate of rHb: A 10.0 μM rHb solution was diluted in 0.2 M potassium phosphate (pH 7.0) buffer. Deoxyhemoglobin was prepared at a concentration ratio of 1:13 (rHb solution: sodium dithionite solution). The complete spectrum of the 400–700 nm wavelength range was recorded using a microplate reader (BioTek Synergy H1) at 37 °C for 64 hours. The values ​​of oxyhemoglobin and methemoglobin were calculated using multi-component analysis, and the auto-oxidation rate in the percentage of oxidized hemoglobin was fitted to a single exponential expression using nonlinear least squares curve fitting.

[0044] Determination of heme binding rate: 0.13 μM rHb was added to a solution containing 3.5 mL pyridine and 1.5 mL 1 M NaOH, and then mixed with 0.2 M sodium dithionite solution. The characteristic absorption peak at 410 nm was immediately detected, and the molar ratio of heme to rHb (i.e., the molar percentage of heme bound per mole of rHb) was determined based on the extinction coefficient to characterize the heme binding rate.

[0045] ε 410 =342500M -1 cm -1

[0046] Determination of heme loss rate: 0.45 M sucrose was added to 0.1 M PBS buffer (pH 7.4), and 6.5 μM methemoglobin was mixed with 40.0 μM sperm whale apomyoglobin mutant apoMb. H64Y / V68F Mixed. Where apoMb H64Y / V68F As a high-affinity heme scavenger, high-concentration sucrose was used as an osmotic agent to prevent the precipitation of apohemoglobin. Absorbance in the 400-650 nm wavelength range was recorded for 4 hours at 37°C. Based on A... 410 The rate of heme loss is calculated using a specific absorbance and a double-exponential expression fitted by nonlinear least squares curve fitting.

[0047] Example 1: Construction of a high-throughput detection strategy for human hemoglobin structural stability mutants

[0048] The structural stability of human hemoglobin is mainly reflected in the fact that structurally stable hemoglobin mutants are less prone to cleavage during intracellular synthesis, which is beneficial for synthesizing higher yields of the target protein. Truncated siroheme synthase (CysG) A It can convert uroporphyrin III to proporphyrin-2, thereby emitting a bright red fluorescence in cells. Therefore, through CysG A G364 Site insertion of the target protein, if the target protein is stable, the fusion can continuously emit fluorescence, and if the target protein is unstable, it will be cleaved, resulting in the CysG in the fusion being divided into two segments A cannot be normally combined, thereby losing the fluorescence signal. Based on this principle, the purpose of high-throughput screening of stable protein mutants can be achieved. The CysG A gene (the nucleotide sequence is shown as SEQ ID NO. 8) was amplified from the genome of Escherichia coli BL21 (DE3) strain and cloned downstream of the T7 promoter of the pETDuet-1 vector. The rHb gene (the nucleotide sequence of the alpha globin is shown as SEQ ID NO. 9, and the nucleotide sequence of the beta globin is shown as SEQ ID NO. 10) was inserted after the 364th amino acid (G A ) of CysG 364 site), and linkers of different lengths (5-54 bp) and glycine percentages (33.0-80.0%) were selected (Table 1) to achieve effective connection between the C-terminal of CysG A (nucleotide sequence shown as SEQ ID NO. 8) and the beta globin (nucleotide sequence shown as SEQ ID NO. 10), a plurality of rHb-CysG A fusion proteins with the structure of "alpha globin-linker 1-CysG A -linker 2-beta globin" were constructed, wherein linker 1 is (SSG)2, and linker 2 is selected from the linking peptides shown in Table 1.

[0049] Table 1 Linkers of different lengths and glycine percentages for fusion expression of the C-terminal of CysG A and beta globin

[0050]

[0051]

[0052] The constructed rHb-CysG A fusion proteins were respectively expressed in BL21 (DE3) strain with pETDuet-1 as the carrier. The constructed recombinant Escherichia coli was cultured in Luria-Bertani medium (0.5% yeast extract, 1.0% peptone, 1.0% sodium chloride, 100 mg / L ampicillin) at 37°C (220 rpm) to OD 600The value reached 0.6, the strain was induced with 10.0 μM IPTG, and 20.0 mg / L of hemin was supplemented simultaneously to promote the synthesis of rHb, the strain was further cultured at 30°C for 10 hours (220 rpm), and the cells were washed twice with 20.0 mM PBS buffer (pH 7.4), and the fluorescence value of CysG A was measured using a microplate reader (BIO-RAD) at a specific wavelength (excitation wavelength 357 nm, emission wavelength 620 nm). The fusion protein rHb-CysG A with the lowest fluorescence intensity was selected A (G4S)3 rHb-CysG

[0053] was selected as a candidate for rHb stability modification A A fusion protein with the structure of “α-globin mutant-(SSG)2-CysG A -(G4S)3-β-globin mutant” was constructed by Gibson assembly method, wherein the rHb mutant was selected from six rHb mutants disclosed in the prior art, including three mutants with better stability (α-G15A, α-G15A / β-G16A and α-G15A / β-G16A / β-H116I) and three mutants with poor stability (α-P119S, α-L29W and β-L28W). The stability mutation energy of the six mutants was calculated based on the Calculate Mutation Energy (Stability) module of the molecular simulation tool Discovery Studio 2019 (α-P119S>α-L29W>β-L28W>wild-type>α-G15A>α-G15A / β-G16A>α-G15A / β-G16A / β-H116I). In addition, according to the CysG A fluorescence value detection method of the rHb-CysG A (G4S)3 fusion protein, the fluorescence intensity of the six rHb mutant-CysG A fusion proteins was determined, and according to the correlation between the fluorescence intensity of the rHb mutant-CysG A fusion protein and the stability mutation energy of the rHb mutant, the reliability of the folding sensor rHb-CysG A (G4S)3 was verified. As can be seen from the results, the correlation of rHb-CysG A (G4S)2 (R2=0.95, P<0.05) was significantly higher than that of the control rHb-CysG Figure 1 (R2=0.72, P>0.05, Figure 1 ), indicating that the folding sensor rHb-CysGA (G4S)3 can be applied to the screening of hemoglobin structure stability mutants.

[0054] Example 2 Construction of high-throughput detection strategy for human hemoglobin heme binding enhanced mutants

[0055] Due to the trace amount of heme binding and the extremely rapid loss of heme during the synthesis of rHb, the current methods for enhancing heme binding cannot meet the demand of high-throughput screening. Therefore, a unique dual-plasmid screening system (rHb-HS1 M7A ) is designed in this example. The first recombinant plasmid is used for the expression of heme detection element HS1 M7A , thereby detecting the escape rate of heme in hemoglobin. The plasmid backbone is pTf16, the screening marker is chloramphenicol resistance, the replicon is RSFori / p15Aori, and the heme detection element HS1 M7A is constructed downstream of the araB promoter and induced to express by arabinose. The heme detection element HS1 M7A is composed of a heme-sensitive fluorescent protein eGFP and a heme-insensitive fluorescent protein mKATE 2, wherein the heme binding domain of cytochrome b 562 in eGFP reduces the fluorescence intensity of eGFP when it binds to heme. The second recombinant plasmid is used for the expression of hemoglobin, the plasmid backbone is pETDuet-1, the screening marker is ampicillin resistance, the replicon is pBR322 ori, and the hemoglobin coding gene is constructed downstream of the T7 promoter and induced to express by IPTG. The recombinant plasmid pTf16-HS1 M7A and the recombinant plasmid pET-rHb carrying the hemoglobin coding gene are co-transformed into the prior art disclosed hemoglobin supply enhanced E. coli HEME-R11 strain (disclosed in the paper Whole-cell P450 biocatalysis using engineered with fine-tuned heme biosynthesis) to obtain a strain with a corresponding dual-plasmid expression system.

[0056] According to the above strategy, the strain rHb wild-type containing the recombinant plasmid pET-rHb wild-type carrying rHb wild-type (encoding gene sequence as shown in SEQ ID NO. 9 and SEQ ID NO. 10) and the strain rHb β-K82D containing the recombinant plasmid pET-rHbβ-K82D strain rHb β-K82D The strain rHb wild-type and strain rHb β-K82D In TB medium (50 mg / L ampicillin and 17 mg / L chloramphenicol), respectively, were cultured at 37°C and 220 rpm until OD reached. 600 When the value reached 0.6, the cells were induced with 40 mM IPTG and 10 mM arabinose at the final concentration. The strain was cultured at 30°C for another 5 hours, then washed twice with 20.0 mM PBS buffer (pH 7.4). The fluorescence ratio of eGFP / mKATE2 in these cells was measured (eGFP: excitation wavelength 488 nm, emission wavelength 523 nm; mKATE2: excitation wavelength 588 nm, emission wavelength 620 nm). rHb-HS1 M7A The eGFP / mKATE2 fluorescence ratio of (RSF ori) reached 4.52 ( Figure 2 This indicates that the system's response range to heme has been significantly enhanced.

[0057] In addition, to accelerate the dissociation rate of rHb-bound heme, rHb-HS1 expression was introduced into the cells. M7A Different heme dissociation reagents were added to 2 mL of (RSF ori) fermentation broth, including cuprous chloride (1.0, 10.0, 100.0, 1000.0 μM), hexadecyltrimethylammonium bromide (2.0, 20.0, 200.0, 2000.0 μM), sodium dodecyl sulfate (50.0, 500.0, 2000.0 μM), and acidified ethanol (0.05%, 0.25%, 5% v / v). After culturing at 30°C for 8 hours (220 rpm), the fluorescence ratio of eGFP / mKATE2 in these cells was detected. The results showed that when 50.0 μM sodium dodecyl sulfate was added, rHb... wild-type and the heme-binding enhanced mutant rHb disclosed in existing technology β-K82D The eGFP / mKATE2 fluorescence ratios decreased by 39.8±2.5% and 41.9±4.0%, respectively, indicating that this concentration of sodium dodecyl sulfate can accelerate the dissociation rate of heme in rHb.

[0058] Finally, to verify rHb-HS1 M7Ascreening efficiency, seven reported rHb mutants (the starting sequences of rHb mutants are shown in SEQ ID NO. 1 and SEQ ID NO. 2) were selected, including five mutants with enhanced heme binding ability (β-V67T, β-F41Y, β-T84Y, β-K82D and β-S44H) and two mutants with reduced heme binding ability (β-H92Q and α-H58L). Using the molecular simulation tool Discovery Studio 2019, the correlation between the fluorescence ratio of eGFP / mKATE2 and the binding mutation energy of these mutants was studied. The results showed that the correlation between the fluorescence ratio of eGFP / mKATE2 and the binding mutation energy of rHb-HS1 M7A (RSF ori) reached 0.89 (P < 0.05), indicating that rHb-HS1 M7A (RSF ori) can be effectively used to screen rHb mutants with enhanced heme binding ability.

[0059] Example 3 Screening of structurally stable human hemoglobin mutants using a semi-rational design strategy

[0060] Based on the structural characteristics of rHb (the coding gene sequences are shown in SEQ ID NO. 9 and SEQ ID NO. 10), appropriate mutation sites were determined by a semi-rational design strategy. The rHb mutation sites were mainly selected from four aspects: (1) subunit contact at the α1β1 interface (34 amino acid residues) and the α1β2 interface (19 amino acid residues); (2) surface amino acid sites of rHb (33 amino acid residues in α-globin and 30 amino acid residues in β-globin); (3) amino acid residues with a B-factor value higher than 40 (possible unstable sites, 8 sites); (4) potential amino acid sites for improving the stability of rHb screened by the virtual alanine scanning module of Discovery Studio 2019 (Stability Mutation Energy <-0.40 kcal / mol, 15 residues). Among these residues, first, sites with specific functions were removed, including the Bohr effect (α-H45, β-L141 and β-H146) and allosteric functions (α-K99, α-R141), etc. Second, 31 key residues that could potentially improve stability (Stability Mutation Energy <-2.00 kcal / mol) were determined by virtual saturation mutagenesis. In order to further improve the screening efficiency, a saturation mutation library was constructed for the 31 key residues of rHb using NNK degenerate bases, and the CysG A fluorescent signal detection of the CysG A fluorescent signal detection of the CysG AThe fluorescence was verified. Based on the ratio of CysG wild-type fluorescence intensity between the mutant and the control (rHb A ), 13 hot-spot residues related to the stability of rHb were determined, which were α-G15, α-K16, α-G25, α-G57, α-V93, α-D94, α-P114, β-H2, β-G16, β-G69, β-S72, β-H97 and β-N102, respectively. These residues could be used for subsequent iterative saturation mutation. In addition, the CysG 1st fluorescence intensity of α-P114I (rHb A ) was the highest, which was 1.75 times of that of rHb wild-type , indicating that α-P114I could be used as the starting mutant for subsequent iterative saturation mutation. Figure 3

[0061] In order to effectively screen the ideal mutant, first, the HbVar database (http: / / globin.bx.psu.edu / hbvar / ) was used to remove the negative mutants that reduced the stability, including rHb (α-G25D) and (β-H97P) mutants. The above 13 hot-spot residues were subjected to saturation mutagenesis using NNK degenerate base pairs, and a plasmid pETDuet-1 carrying rHb mutants was constructed, in which the starting sequence of hemoglobin mutants (the nucleotide sequence of α-globin is shown as SEQ ID NO. 9, and the nucleotide sequence of β-globin is shown as SEQ ID NO. 10) was constructed downstream of the T7 promoter. The plasmid carrying the rHb mutant was transformed into the BL21 (DE3) strain, and 100 clones were screened for each amino acid residue site to achieve 95% coverage in the library. Each single colony was further transferred to a 96-well plate containing 0.2 mL Luria-Bertani medium (100 mg / L ampicillin), and cultured at 37°C (220 rpm) to an OD 600 value of 0.6. The strain was induced with 10.0 μM IPTG, and 20.0 mg / L hematin was supplemented at the same time to promote the synthesis of rHb. The strain was cultured at 30°C for 10 hours (220 rpm), and the cells were washed twice with 20.0 mM PBS buffer (pH 7.4). The CysG A fluorescence value of these cells was measured, and the mutant with the highest fluorescence value in each round of iterative saturation mutagenesis was selected for the next round of saturation mutagenesis. After six rounds of iterative saturation, the CysG 6th fluorescence intensity of rHb A (α-G25C / G57K / P114I, β-H2E / H97W / N102T) was improved by 7.87 times compared with rHb wild-type , and the ideal mutant rHb​5th (α-G25C / G57K / P114I, β-H2E / N102T) CysG A The similar fluorescence values ​​indicate that an ideal stable rHb mutant has been obtained. 6th Meanwhile, the oxygen affinity of all ideal mutants in each iteration was examined to ensure their future application in the field of artificial oxygen carriers.

[0062] To verify rHb 6th The stability of rHb was assessed by examining its secondary structure, thermal stability, and autoxidation rate. The secondary structure of rHb was verified using the circular dichroism signal at 222 nm, and guanidine hydrochloride (GdnHCl) was used as the denaturant for rHb. 6th [GdnHCl] midpoint The concentration of GdnHCl (at 50% secondary structure dissociation) increased to 1.90 M, compared to rHb. wild-type (1.50M) increased by 26.7%. Furthermore, rHb 6th The melting temperature (TM) increased to 69.74 ± 0.05 °C, compared to rHb. wild-type (64.04±0.13℃) ​​increased by 8.9%. In addition, rHb 6th auto-oxidation rate (k autox =0.011±0.005 / h) ratio to rHb wild-type (k autox =0.167±0.034 / h) decreased by 14.83 times, indicating that rHb 6th It exhibits good resistance to auto-oxidation. Furthermore, due to improved stability, rHb... 6th The yield (83.23±1.51 mg / L) is rHb wild-type 1.51 times that of rHb. Therefore, compared to rHb... wild-type In comparison, rHb 6th It offers significant advantages in commercial production and application, including higher stability, antioxidant properties, and yield.

[0063] Example 4: Screening for heme-binding enhanced human hemoglobin mutants using a semi-rational design strategy.

[0064] rHb-HS1 constructed in Example 2 M7A The (RSF ori) system can be used to screen rHb mutants with enhanced heme-binding ability. To determine suitable modification sites through semi-rational design, rHb... 6th Around the heme pockets in α and β globin residues in the CE helix that affect heme binding in the b-globin protein were virtually saturated. Then, based on the mutations in the virtual saturation that significantly enhanced the heme affinity (Binding Mutation Energy <-0.60 kcal / mol), 26 sites were selected as candidate sites. Subsequently, to narrow the screening range, the 26 candidate residues were saturated with NNK degenerate bases, and plasmids carrying rHb mutants were transformed into E. coli R11-HEME strain, and at least 100 clones were selected for each key site to verify the effect of the mutation. According to the increase in the eGFP / mKATE2 fluorescence ratio (>1.50-fold) between the mutants and the control (rHb 6th ), 9 sites were further determined for subsequent iterative saturation mutation, which were a-F33, a-Y42, a-F43, b-W15, b-S44, b-P51, b-K66, b-K82 and b-D99.

[0065] To efficiently obtain the desired mutants, the above 9 sites were saturated with NNK degenerate bases, and plasmids carrying rHb mutants pETDuet-1 were constructed, in which the coding gene (nucleotide sequence as shown in SEQ ID NO. 9 and SEQ ID NO. 10) of the hemoglobin mutant (amino acid sequence as shown in SEQ ID NO. 1 and SEQ ID NO. 2) was constructed downstream of the T7 promoter and expressed by IPTG induction. The pETDuet-1 plasmid carrying the rHb mutant and the pTf16 plasmid carrying the HS1 M7A strain, and 100 clones were selected for each amino acid residue site to achieve 95% coverage in the library. Each single colony was further transferred to a 96-well plate containing 0.2 mL TB medium (50 mg / L ampicillin, 17 mg / L chloramphenicol), and cultured at 37°C, 220 rpm to an OD 600 value of 0.6. The strain was induced with 40 mM IPTG and 10 mM arabinose and cultured at 30°C, 220 rpm for 5 hours. Subsequently, 0.05 mM SDS was added to the fermentation broth to accelerate the loss rate of heme in rHb. The strain was further cultured at 30°C, 220 rpm for 8 hours, and the bacterial cells were collected by centrifugation and washed twice with 20.0 mM PBS buffer (pH 7.4). The eGFP / mKATE2 fluorescence ratio of these cells was measured, and the mutant with the highest fluorescence ratio in each round of iterative saturation mutagenesis was selected for the next round of saturation mutagenesis. After three rounds of iterative saturation mutation, rHb 9thThe eGFP / mKATE2 fluorescence ratio of (α-G25C / Y42S / G57K / P114I, β-H2E / S44D / P51N / H97W / N102T) compared to rHb 6th Increased by 2.64 times ( Figure 4 In addition, rHb 9th CysG A Fluorescence intensity ratio rHb 6th The 3.6% increase indicates that subsequent mutations did not affect rHb. 6th The stability of rHb 9th It is an ideal mutant with a significantly enhanced ability to bind heme.

[0066] Finally, due to rHb 9th The heme pocket region in the heme cells underwent changes, resulting in a significant decrease in the rate of heme loss (k). -H β=1.32±0.10 / h,k -H α=0.02±0.004 / h), and rHb 6th In comparison, rHb 9th The heme dissociation rate of β-globin decreased by 1.40-fold, and that of α-globin decreased by 6.00-fold. rHb 9th The enhanced heme-binding capacity also helped increase rHb production (84.94±2.70 mg / L) and heme binding rate (55.7±1.8% mol heme / mol rHb), and reduced the auto-oxidation rate (kJ / mol rHb). autox =0.008±0.003 / h), while rHb 9th With rHb 6th There were no significant differences in other properties.

[0067] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A hemoglobin mutant, characterized in that, Based on the starting sequence, perform the mutation shown in (a) or (b): (a) Mutate glycine at position 25 of α-globin to cysteine, glycine at position 57 to lysine, and proline at position 114 to isoleucine; mutate histidine at position 2 of β-globin to glutamic acid, histidine at position 97 to tryptophan, and asparagine at position 102 to threonine. (b) Mutate glycine at position 25 of α-globin to cysteine, tyrosine at position 42 to serine, glycine at position 57 to lysine, and proline at position 114 to isoleucine; mutate histidine at position 2 of β-globin to glutamic acid, serine at position 44 to aspartic acid, proline at position 51 to asparagine, histidine at position 97 to tryptophan, and asparagine at position 102 to threonine; The starting sequence of α-globin is shown in SEQ ID NO.1; the starting sequence of β-globin is shown in SEQ ID NO.

2.

2. The gene encoding the hemoglobin mutant of claim 1.

3. A recombinant plasmid carrying the gene described in claim 2.

4. A recombinant microorganism expressing the hemoglobin mutant of claim 1.

5. Recombinant Escherichia coli, characterized in that, The hemoglobin mutant of claim 1 was expressed using pETDuet-1 as an expression vector.

6. A method for preparing hemoglobin, characterized in that, The recombinant microorganism of claim 4 or the recombinant Escherichia coli of claim 5 is cultured in a culture medium.

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